Methods of evaluating a test agent in a diseased cell model

ABSTRACT

The present invention relates to methods of constructing an integrated artificial immune system that comprises appropriate in vitro cellular and tissue constructs or their equivalents to mimic the tissues of the immune system in mammals. The artificial immune system can be used to test the efficacy of vaccine candidates and other materials in vitro and thus, is useful to accelerate vaccine development and testing drug and chemical interactions with the immune system, coupled with disease models to provide a more complete representation of an immune response.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.12/833,640, filed Jul. 9, 2010, now issued as U.S. Pat. No. 8,062,889,which is a divisional of U.S. application Ser. No. 12/047,107, filedMar. 12, 2008, now issued as U.S. Pat. No. 7,771,999, which was acontinuation-in-part of U.S. application Ser. No. 11/453,003, filed Jun.15, 2006, now issued as U.S. Pat. No. 7,709,256, which was acontinuation-in-part of U.S. application Ser. No. 11/116,234, filed Apr.28, 2005, now issued as U.S. Pat. No. 7,855,074, which claims thebenefit of priority of both U.S. Provisional Application Ser. No.60/565,846, filed Apr. 28, 2004, and U.S. Provisional Application Ser.No. 60/643,175, filed Jan. 13, 2005. Each of these applications ishereby incorporated by reference in its entirety.

STATEMENT REGARDING FEDERALLY. SPONSORED RESEARCH

This invention was made with government support under contract numberNBCHC060058, awarded by the Defense Advanced Research Projects Agency,issued by the U.S. Army Medical Research Acquisition Activity, andadministered by the U.S. Department of the Interior-National BusinessCenter. The government has certain rights in the invention.

FIELD OF THE INVENTION

The present invention is directed to a method for developing a diseasemodel that may be used with an artificial human immune system for invitro testing of vaccines, adjuvants, immunotherapy candidates,cosmetics, drugs, biologics, and other chemicals. The disease model andartificial immune system of the present invention is useful forassessing disease pathogenesis and the effect of vaccines, drugs,biologics, immunotherapeutics, and adjuvants on a disease. In thecontext of vaccines, for example, the disease models and artificialimmune system of the present invention could be used to predict theeffectiveness of a vaccine by means of an in vitro challenge withdisease agents. The disease models and artificial immune systems of thepresent invention can be prepared using cells from healthy (notdiseased, uninfected, naïve) individuals or from individuals sufferingfrom diseases or infections. “Diseased cells” include virally infectedcells, bacterially infected cells, and tumor cells, and cells andtissues affected by a pathogen or involved in an immune-mediateddisease, such as, e.g., autoimmune disease. Embodiments of the presentinvention can be used to accelerate and improve the accuracy andpredictability of vaccine and drug development.

BACKGROUND OF THE TECHNOLOGY

The development and biological testing of human drugs and vaccines hastraditionally relied on small animal models (e.g., mouse and rabbitmodels) in the early stages and then on larger animals, such as dogs andnon-human primates, in later stages. However, animal models of diseaseare often only approximations of the human disease state, and in somecases animal models are not available at all (e.g., pathogens thatinfect only humans). Thus, animal models of disease may not accuratelypredict outcomes in human studies or may not be available to make suchpredictions.

In the case of diseases that involve human immunology, such as an immuneresponse to a pathogen or a deleterious inflammatory response, likepsoriasis, a major problem remains the translation from animal testsystems to human immunology. Successful transfer between traditionaltesting systems and human biology requires an intricate understanding ofdisease pathogenesis and immunological responses at all levels. Thus,there is a need for a system that uses human immune cells to simulatehuman immune responses in the context of a disease state.

SUMMARY OF THE INVENTION

The present invention comprises the use of an artificial immune system(AIS) with disease models to provide essentially the ability to conducta “clinical trial in a test tube” to predict the efficacy of a vaccine,adjuvant, drug, or other agent on a disease that involves the immunesystem (e.g., pathogen response, autoimmune disease, cancer response).FIG. 1 illustrates schematically an example of the integration of theAIS and a disease model. As an example, schematically, the VS is whereinfection or vaccination occurs, the LTE is where immune inductionoccurs, and the disease model is where the immune response to thedisease occurs.

Models of the present invention are particularly appropriate forexamining diseases and infections that involve immune system cells.Examples include HIV, tuberculosis, tularemia, filoviruses, Yersinia,and Burkholderia. The in vitro disease models of the present inventioncan also be used to understand basic disease pathogenesis. Theartificial immune system of the present invention comprises threemodules: the first simulates the innate immune response (the vaccinationsite, VS, module), the second simulates the adaptive immune response forthe detection of T and B cell responses in vitro, providing a model forthe interaction of immune cells within lymph nodes (the lymphoid tissueequivalent, LTE, module); and the third module is a functional assay ordisease module that uses the products of the other two modules togetherwith, for example, a pathogen or human tissue, to measure the effect ofthe immune response to a disease, such as influenza, glanders,tularemia, tuberculosis, Ebola, Marburg, plague, or AIDS, directlyrather than through the use of surrogate markers. These artificialimmune system modules reproduce the conditions that exist in the humanbody, such as the spatial segregation of different immune cells andtemporal dynamics that bring different immune cells together atdifferent times. Other variations are possible, such as using the LTEfor both immune induction and the immune response, so that the LTEeffectively becomes a disease model.

The disease models and artificial immune systems of the presentinvention can be prepared using cells from healthy (not diseased,uninfected, naïve) individuals or from individuals suffering fromdiseases or infections. “Diseased cells” include pathogen-infectedcells, e.g., virally infected cells and bacterially infected cells,tumor cells, and cells and tissues affected by a pathogen or involved inan immune-mediated disease, such as, e.g., autoimmune disease.

As examples, disease models of the present invention include viral(e.g., herpes simplex virus, hepatitis A, B, C, VSV, HIV, vacciniavirus, influenza virus), tumoral (e.g., melanoma), and autoimmune models(e.g., RA, diabetes, psoriasis, Crohn's disease).

A primary goal of a preclinical testing program is to improve outcomefor patients by the early identification of potential applications fornew vaccine or drug agents before clinical development. The premise forestablishing an in vitro testing effort is that it will allow candidatesto be selected for clinical evaluation with increased likelihood forclinical benefit. Clearly, this requires that the in vitro system bepredictive of human responses to the vaccine or drug and the efficacy ofthe vaccine or drug against the disease in question. In the absence ofan effective and predictive preclinical testing program, ineffectivevaccines and drugs are likely to be selected for evaluation, thusslowing progress in improving outcomes. Furthermore, having an in vitrotesting system that is predictive (a “clinical trial in a test tube”)will significantly reduce lost opportunity costs associated with vaccineor drug testing. That is, if a candidate is going to fail, it shouldfail early.

The development of an artificial immune system coupled with a diseasemodel has the potential to change the way vaccines and drugs are tested.The preclinical in vitro testing program of the present invention,though based on both immunologic and engineering principles, has thevery pragmatic objective of providing reliable, predictive, andreproducible information to clinical investigators to allow enlightenedprioritization among the multiple candidates available. Clearly, that isa goal of all preclinical testing, but what is new in the in vitrotesting system of the present invention is an in vitro model usingfunctionally equivalent tissue engineered constructs populated withhuman cells. In comparison with in vivo animal testing, in vitro testingusing the system comprising the present invention is less expensive,less time-consuming, and importantly more predictive of clinicaloutcomes.

The present invention concerns the development of accurate, predictivein vitro models to accelerate vaccine testing, allowing collection ofmore informative data that will aid in redesigning and optimizingvaccine and drug formulations before animal or clinical trials, andraise the probability that a vaccine or drug candidate will besuccessful in human trials.

More specifically, the present invention comprises controlling thenature and state of the cells in the lymphoid tissue equivalent (LTE,artificial lymph node) of the artificial immune system (AIS). The AIScan be used to test vaccines and other pharmaceuticals for immunereactivity in a manner that is more predictive than animal experiments.Consequently, it can provide valuable pre-clinical data earlier in theresearch and development process. Antigenic molecules introduced to theAIS are acquired by dendritic cells (DCs) at the vaccination site (VS).The DCs are then transferred to the lymphoid tissue equivalent (LTE),where they present the antigen to T cells, activating their immunefunction. Activated helper T cells co-stimulate B cells to induceantibody production, while activated cytotoxic T cells lyseantigen-bearing cells. Solubilized antigen(s) can also be introducedinto the LTE to directly activate B cells for subsequent antibodyproduction. In other embodiments, pathogens, not antigenic molecules,are introduced to the AIS and infect APCs, e.g., dendritic cells (DCs),at the vaccination site (VS). In other embodiments, pathogens areintroduced to the AIS and infect cells in the LTE.

While a number of published reports have demonstrated antigen-specific Bcell responses (to C. albicans, TT, and other antigens) in vitro, theseresults are typically achieved by stimulating and restimulating culturesof whole PBMCs with antigen and exogenous factors to boost B cellproliferation and/or activation. Embodiments of the present inventioncomprise the detection of immune responses using defined cultures of Bcells, T cells, and DCs and optionally follicular dendritic cells(FDCs), in 2-dimensional construct assays. The presence of secondarycells provides a more physiological environment for B cell activationand differentiation, such that artificial factors in the cultures arenot necessary to detect specific immune responses.

Using embodiments of the present invention, we have generatedantigen-specific B cell responses using a (2D) co-culture systemcomprising T cells, B cells, and antigen-pulsed DCs. Responses have beengenerated against tetanus toxoid (TT) and a whole protein extract ofCandida albicans (C. albicans) influenza, recombinant protective antigenof Bacillus anthracis, hepatitis B, yellow fever, rabies and merozoitesurface protein 1 (MSP-1) from malaria. These results show thatculturing human T and B cells together in vitro at, for example, a ˜1:1ratio, versus the ratio of T and B cells naturally found in the blood,gave stronger antigen responses, by both analysis of activation andproliferation (flow cytometry) and antibody production (ELISPOT). Here,“T cells” includes both CD4⁺ and CD8⁺ T cells. In peripheral blood, theT (total T cells):B ratio is ˜7:1. In the lymph node, the T (total Tcells):B ratio is ˜1:1.6. In the germinal center, the T:B ratio is ˜1:8,and there, the T cells are primarily CD4⁺ T cells.

It is known that 3-dimensional biology is important to induce properfunctionality of immunological engineered tissue constructs (ETCs; see,e.g., Edelman & Keefer, Exp. Neurol. 192:1-6 (2005)). A principalapproach to studying cellular processes is to culture cells in vitro.Historically, this has involved plating cells on plastic or glasssupports. Cells grown on solid or filter support are referred astwo-dimensional (2D) cultures. Such 2D cultures on porous supports havebeen useful in studying many aspects of biology. However, more invivo-like conditions can now be realized in 3D cultures.

In lymph nodes, it has been shown that 3D interstitial tissue matrixfacilitates not only T cell migration toward an APC, but also supportsmotility upon cell-cell interaction. A 3D collagen matrix environment,because of its spatial architecture, provides traction for lymphocytecrawling, mimicking some structural features of the lymph node cortex.This provides experimental justification for the importance of a 3Denvironment in the constructs that comprise some embodiments of theartificial immune system of the present invention.

The present invention also differs significantly from existing in vitrodisease models. For example, simple monolayer and suspension culturesare commonly used to model viral infection and tumors. However, suchcell cultures provide a highly artificial cellular environment and arenot coupled to an artificial immune system.

The disease models of the present invention include cancer models.Although historically mice have been used for studying tumor genetics,physiology, and therapeutic regimens, murine tissue models have manylimitations. An important difference is that human tumors are primarilyepithelial in origin, whereas murine tumors are typically non-epithelial(such as sarcomas, lymphomas). Many agents that are carcinogenic in miceare not in humans, and vice versa. Oncogenic pathways are different inmany ways in the mouse compared to humans. Additionally, the murinebasal metabolic rate is six times higher than in humans. New approacheshave examined xenograft placement on immune- deficient mice with moresuccess; however, the murine component still exists in this model.(Ortiz-Urda et al. (2002) Nature Med 8, 1166-70). Thus, studying humantumor models in a human cell-based model of the present inventionremoves these interspecies differences.

Recent work by Mertsching and colleagues at the Fraunhofer Institute ofInterfacial Engineering and Biotechnology, Germany, is beginning todemonstrate that in vitro 3D models can be a useful platform in cancerresearch. They developed a new, 3D, vascularized tissue construct. Thevascularized 3D matrix is populated with endothelial cells and then withtumor cells to create an ex vivo vascularized tumor-like structure as adisease model. Their data suggests that this in vitro model offers thepossibility to simulate physiological drug application and provide ahuman 3D test system for cancer research/therapy.

In the present invention, such a 3D tumor model could be used inconjunction with an artificial immune system to predict theeffectiveness of a cancer vaccine on a tumor rather than on isolatedcancer cells in a 2D culture. This distinction is important. Antibodiesand immune effector cells must reach the tumor in sufficient numbers tohave an impact on the disease. Access to the cancer cells is not anissue with cell cultures, but can become a problem with a vascularizedtumor. In addition, tumors may induce surrounding tissues to secretefactors that induce immune tolerance, thus counteracting the immuneenhancement induced by a cancer vaccine. These types of effects may notbe observed in the absence of a tumor disease model that interacts withan artificial immune system.

The disease models of the present invention include pathogen infectionmodels (e.g., viral, bacterial, fungal, protozoan, parasitic). Inembodiments of the present invention, we use cells in 2D culture. Inother embodiments, we use cells placed with a 3D tissue-engineeredconstruct. The infected or diseased cells can be included in theengineered tissue construct. For example, virally infected epithelialcells can be used in a tissue engineered skin or mucosal equivalent. Asanother example, herpes simplex viruses are ectodermotropic (i.e., theycan infect and reproduce in epithelial cells and reside in neurons in alatent state). The disease model in this case could use virally infectedepithelial cells or neurons, or both, to model both active and latentherpes virus infection. In other embodiments of the present invention,we use engineered tissue constructs to model viral transport andinfection in compartments that are separated from the primary infectionsite.

For example, HIV-1 is captured by dendritic cells (DCs) and delivered tothe lymph node, where the virus is then transmitted to CD4⁺ T cells. Thelymph node then becomes the principal site of virus production. In anembodiment of the present invention, the vaccination site (VS) modulewould be the infection site, where HIV infects the APCs, e.g., DCs,which would then be placed into the lymphoid tissue equivalent module,where the infected DCs transmit the virus to CD4⁺ T cells. In this case,the LTE would serve as the disease module. Collectively, the infectionsite and disease module would comprise the disease model.

The disease models of the present invention include inflammatory andautoimmune diseases. In these diseases (e.g., psoriasis, rheumatoidarthritis), the immune system itself is primarily responsible for thedisease state. In one embodiment of the present invention, an LTE couldbe constructed from immune cells isolated from the blood of donorsafflicted with psoriasis. An engineered tissue construct could be madefrom biopsied skin from the same donor. The interaction of the AIS andthe disease model under different conditions could yield insight intodisease pathogenesis. Also, one could test different candidate drugs forpsoriasis in this disease model to determine potential efficacy of acandidate. If this process was repeated for a large number of donorsafflicted with psoriasis, the resulting “clinical trial in a test tube”could facilitate the selection of an optimal clinical candidate for thelargest number of patients or the selection of several candidates, whichare targeted at different patient populations through the use ofclinically relevant biomarkers. In another embodiment of the presentinvention, the disease model would simulate an aspect of an autoimmuneor inflammatory disease, rather than the disease itself. For example, animportant hallmark of certain inflammatory diseases is the migration ofneutrophils to the site of inflammation. If this process of neutrophilmigration could be interrupted, then the inflammatory process could beinterrupted, with a corresponding beneficial effect on the diseasestate. The AIS could, thus, be used to model neutrophil migration as aproxy for modeling the resulting inflammatory disease.

HIV models. HIV (human immunodeficiency virus) is the virus that causesAIDS (acquired immune deficiency syndrome). An embodiment of the presentinvention comprises a HIV disease model. In vivo, HIV-1 infects DCs andthey move to the lymph nodes where the virus infects CD4⁺ T cells. Thelymph node then becomes the principal site of virus production.

Infection with HIV-1 is associated with a progressive decrease in theCD4⁺ T cell count and an increase in viral load. The stage of infectioncan be determined by measuring the patient's CD4⁺ T cell count, and thelevel of HIV in the blood. This acute viremia is associated in virtuallyall patients with the activation of CD8⁺ T cells, which killHIV-infected cells, and subsequently with antibody production, orseroconversion. The CD8⁺ T cell response is thought to be important incontrolling virus levels, which peak and then decline, as the CD4⁺ Tcell counts rebound to ˜800 cells/mL (normal is ˜1200 cells/mL). Astrong CD8⁺ T cell response has been linked to slower diseaseprogression and a better prognosis, though it does not eliminate thevirus. During this early phase of infection, HIV is active withinlymphoid organs, where large amounts of virus become trapped in thefollicular dendritic cells (FDC) network. The surrounding tissues thatare rich in CD4⁺ T cells may also become infected, and viral particlesaccumulate both in infected cells and as free virus.

Tularemia models. Francisella tularensis is a Category A biowarfareagent and is an important focus of biodefense research. An embodiment ofthe present invention comprises a tularemia disease model. Thepathogenicity of F. tularensis is quite similar to that of Mycobacteriumtuberculosis (Mtb), in that both infect macrophages and dendritic cells(Clemens et al. (2004) Infect. Immun. 72, 3204-17). F. tularensis is aGram-negative bacterium responsible for tularemia, a zoonotic diseasethat affects many mammals and is occasionally transmitted to humansthrough tick bites, by direct contact with an infected animal, orthrough aerosolization of contaminated materials. The progression andthe severity of the disease depend on the host immune status and on theinfecting strain. There are four known subspecies of F. tularensis(tularensis, holarctica, mediasatica, novicida); tularensis is the mostvirulent. The live vaccine strain (LVS) was derived from the holarcticasubspecies and is widely used to study tularaemia. The subspeciesnovicida is less virulent in humans.

F. tularensis infects macrophages, dendritic cells, hepatocytes andalveolar epithelial cells. Its virulence depends on its ability tomultiply inside host cells. Upon entering the host cell, F. tularensisis taken up into a phagosome. It prevents acidification and maturationof the phagosome (Clemens et al. (2004) Infect. Immun. 72, 3204-17),escapes the phagosome, and multiplies in the cytosolic compartment ofthe host cells. This replication is dependent on a cluster of genesknown as the Francisella pathogenicity island (FPI). Upon escaping intothe cytosol, F. tularensis activates many pathways of the innate immunesystem, including the inflammasome, which triggers both acaspase-1-mediated apoptotic cascade in the host cell and also apro-inflammatory cytokine response (Henry & Monack (2007) Cell.Microbiol. 9, 2543-255). Cytokines including IL-18, IL-1b, IFN-γ, IL-12,and the Th2 cytokines IL-4 and IL-5, are known to play important rolesin the immune response against F. tularensis (Henry & Monack (2007)Cell. Microbiol. 9, 2543-255).

Although the role of antibodies in protection against respiratoryinfection with F. tularensis is unclear, one study reported prophylacticand therapeutic use of antibodies for protection against respiratoryinfection (Kirimanjeswara et al. (2007) J. Immunol. 179, 532-9). Serumantibodies (immune serum from infected mice) were capable of conferringcomplete protection against lethal respiratory tularemia to a naïve micewhen given 24-48 h post-exposure.

Filovirus models. The filoviruses Marburg and Ebola are Category Abiowarfare agents. An embodiment of the present invention comprises afilovirus disease model. Filoviruses are enveloped, non-segmented,negative-stranded RNA viruses. The virions contain a ˜19 kbnon-infectious genome that encodes seven structural proteins, with agene order of: 3′ leader, nucleoprotein (NP), virion protein (VP) 35(VP35), VP40, glycoprotein (GP), VP30, VP24, polymerase L protein, and5′ trailer (Sanchez et al. (199) Virus Res. 29, 215-240). Studies usingreconstituted replication systems showed that transcription/replicationof Marburg virus requires three of the four proteins (NP, VP35, L),while transcription/replication of Ebola virus requires all fourproteins (Muhlberger et al. (1999) J. Virol. 73, 2333-2342). GP is thesurface glycoprotein of the virion and is important for receptor bindingand membrane fusion (Takada et al. (1997) Proc. Natl. Acad. Sci. USA 94,14764-69; Ito et al. (1999) J. Virol. 73, 8907-8912).

Most research to date on filovirus infections has come from experimentalinfection of non-human primates, including cynomolgus macaques andAfrican green monkeys. Rodent models of filovirus infection haveprovided data on the efficacy of candidate drugs and vaccines, but donot faithfully reproduce the viral pathogenesis and immunity in humans(Bray et al. (2001) J. Comp. Pathol. 125, 243-253; Geisbert et al.(2002) Emerg. Infect. Dis. 8, 503-507). Indeed, there is currently onlylimited data available on the pathophysiology of filovirus infections inhumans.

Both Ebola and Marburg viruses have broad cell tropisms and use avariety of host cell surface molecules to gain entry into host cells.Infection of monocytes/macrophages and dendritic cells is central to thepathology of Ebola virus infection (Stroher et al. (2001) J. Virol. 75,11025-33), triggering a cascade of events leading to the production andrelease of the procoagulant protein tissue factor (TF) (Geisbert et al.(2003a) J. Infect. Dis. 188, 1618-1629) and a variety ofcytokines/chemokines (Stroher et al. (2001) J. Virol. 75, 11025-33;Hensley et al. (2002) Immunol. Lett, 2002, 80, 169-179). Althoughlymphocytes are not infected by Ebola or Marburg viruses, there islarge-scale destruction of these cells, said to be the result of“bystander” apoptosis (Geisbert et al. (2000) Lab. Invest. 80, 171-86).Reasons for this aberrant apoptosis of lymphocytes are unclear. Anembodiment of the present invention comprises an in vitro disease modelthat can be used to explore this phenomenon.

It has been suggested that infection of monocytes/macrophages, damage toendothelial cells, and release of procoagulant protein tissue factorappear to be the cause for the development of hemorrhage, shock, andcoagulation defects such as disseminated intravascular coagulation (DIC)during filoviral infections (Geisbert et al. (2003a) J. Infect. Dis.188, 1618-1629).

In studies involving infection of non-human primates with Ebola virus,increased circulating levels of IFNa, IL-6, MCP-1, MIP-1a, MIP-1b,IFN-β, IFN-γ, IL-18, and TNF-α at different stages of disease wereobserved (Geisbert et al. (2003b) Am. J. Pathol. 163, 2347-2370). Inhuman cases, an association between increased levels of IL-10 andincreased fatalities was observed in Ebola virus infection (Baize et al.(2002) Clin. Exp. Immunol. 128,163-168). Other in vitro studies withdifferent types of primary human cells showed similar increases inproinflammatory cytokine levels (Stroher et al. (2001) J. Virol. 75,11025-33; Hensley 2002) and also that results varied due to humandonor-associated genetic differences. In using the VS module to modelfilovirus disease, cytokine production in the VS module is monitored asare phenotypic changes in the APCs.

Early attempts to develop filoviral vaccines have used cellculture-propagated filoviruses inactivated with formalin or heattreatment, but the protection offered was inadequate (Geisbert et al.(2002) Emerg. Infect. Dis. 8, 503-507). Current efforts use differentrecombinant vectors, such as VSV and adenoviral vectors, for expressionof filoviral-encoded proteins individually or in combinations (Geisbert& Jahrling (2003c) Exp. Rev. Vaccines 2, 777-789). GP and NP are beingtested as vaccine candidates in non-human primates with encouragingresults (Sullivan et al. (2003) Nature 424, 681-4). There are currentlyno effective post-exposure treatments for filoviral infections.

Yersinia models. The genus Yersinia includes three species that arepathogenic to humans, Y. enterocolitica, Y. pestis, and Y.pseudotuberculosis (Brubaker (1991) Clin. Microbiol. Rev. 4, 309-324).An embodiment of the present invention comprises a Yersinia diseasemodel. Y. pestis, a Gram-negative bacterium, is the agent of plague, alethal disease transmitted by flea bites or by aerosols (Perry &Fetherston (1997) Clin. Microbiol. Rev. 10, 35-66). Y. pestis has beenthe cause of three pandemics (Drancourt et al. (2004) Emerg. Infect.Dis. 10, 1585-92), and has resulted in the deaths of millions of people.

Y. pseudotuberculosis and Y. pestis are closely related. It is believedthat Y. pestis may have evolved from Y. pseudotuberculosis (Skurnik etal. (2000) Mol. Microbiol. 37, 316-330; Achtman et al. (1999) Proc.Natl. Acad. Sci. USA 96, 14043-48). Minor phenotypic differences havebeen used to classify Y. pestis strains into three biovars (Antigua,Mediaevalis, Orientalis) (Perry & Fetherston (1997) Clin. Microbiol.Rev. 10, 35-66). Despite differences in their mode of entry into thehost and severity of disease, all three pathogenic Yersinia speciesexhibit a common tropism for lymphoid tissue (Brubaker (1991) Clin.Microbiol. Rev. 4, 309-324). The complete genome sequence of Y. pestishas been determined (Parkhill et al. (2001) Nature 413, 523-527; Deng Wet. al. (2002) J. Bacteriol. 184, 4601-4611).

In the pathogenic Yersinia spp., several virulence factors have beenidentified that promote serum resistance and the acquisition of iron(Brubaker (1991) Clin. Microbiol. Rev. 4, 309-324; Perry & Fetherston(1997) Clin. Microbiol. Rev. 10, 35-66; Camiel 2002). Additionally, theycontain a 70-kb plasmid that is necessary for sustained bacterialreplication in host tissues (Cornelis et al. (1998) Microbiol. Mol.Biol. Rev. 62, 1315-1352). A type III secretion system (TTSS) andseveral secretion substrates (Yops, LcrV) are expressed from thevirulence plasmid when Yersinia spp. are grown at 37° C. (Cornelis etal. (1998) Microbiol. Mol. Biol. Rev. 62, 1315-1352; Perry & Fetherston(1997) Clin. Microbiol. Rev. 10, 35-66). After they are secreted by theTTSS, the Yops are delivered into the phagocytes, where they inhibitphagocytosis and proinflammatory cytokine production and also triggerapoptosis of host cells (Cornelis (2002) J. Cell. Biol. 158, 401-408).LcrV is secreted into the extracellular milieu where it inhibitsinflammation by interacting with Toll-like receptor 2 (Brubaker (2003)Infect. Immun. 71, 3673-3681). Y. pestis carries two plasmids, pMT1 andpPCP1, that impart Y. pestis with increased virulence (pMT1 and pPCP1)and vector-borne transmissibility (pMT1) (Carniel (2002) Cum Top.Microbiol. Immunol. 264, 89-108; Perry & Fetherston (1997) Clin.Microbiol. Rev. 10, 35-66).

Y. pestis causes bubonic plague when its mode of entry is intradermalfollowing the bite from an infected flea, while it causes pneumonicplague when infection is by inhalation of infectious droplets (Brubaker(1991) Clin. Microbiol. Rev. 4, 309-324; Perry & Fetherston (1997) Clin.Microbiol. Rev. 10, 35-66). Disease pathogenesis has been studied inmice and non-human primates (Welkos et al. (1997) Microb. Pathogen. 23,211-223; Finegold (1969) Am. J. Pathol. 54, 167-185). Bacteria multiplyat the initial site of infection before entering the lymphatic systemand spread to regional lymph nodes and via the bloodstream to otherorgans, such as spleen and liver. Macrophages may act as the vehicle fortransport from the initial site of infection to the lymphoid tissues.Extensive bacterial replication in visceral organs leads to septicemiaand death of the host (Brubaker (1991) Clin. Microbiol. Rev. 4, 309-324;Perry & Fetherston (1997) Clin. Microbiol. Rev. 10, 35-66). Animalstudies have indicated that in the later stages of infection process(more than 12 h post-infection) Y. pestis was found to replicate innecrotic foci extracellularly (Welkos et al. (1997) Microb. Pathogen.23, 211-223; Nakajima et al. (1995) Infect. Immun. 63, 3021-3029).

Y. pestis has long been considered a facultative intracellular pathogen(Cavanaugh & Randall (1959) J. Immunol. 85, 348-363). Animal studieshave indicated that Y. pestis can survive and replicate withinmacrophages (Finegold 1969), but is killed intracellularly byneutrophils (Cavanaugh & Randall (1959) J. Immunol. 85, 348-363; Burrows& Bacon (1956) Br. J. Exp. Pathol. 37, 481-493). Thus, macrophageseffectively serve as permissive sites for replication in the earlystages of infection. Y. pestis achieves this by subverting the normalantibacterial functions of macrophages. A study in inbred and outbredstrains of mice infected with pneumonic plague showed thatproinflammatory cytokines, such as like IL-6, TNFα, IFNγ, IL-12, andMCP-1 are found in the bronchoalveolar lavage fluids in later stages ofinfection (Bubeck et al. (2007) Infect. Immun. 75, 697-705). In the hostimmune response to plague, Y. pestis-infected human monocytes werereported to express TLR9 and differentiate into dendritic cells (Saikhet al. (2004) J. Immunol. 173, 7426-34). Y. pestis is known to evadeimmune responses in part by injecting host immune cells with severaleffector proteins called Yersinia outer proteins (Yops) that impaircellular function. Y. pestis YopJ disrupts signal transduction pathwaysand interferes with DC differentiation and subsequent function (Lindneret al. (2007) Eur. J. Immunol. 37, 2450-62). Further, YopJ injectionprevents upregulation of costimulatory ligands, and LPS-induced cytokineexpression in DC thus crippling the adaptive response via a diminishedcapacity to induce T cell proliferation and IFNγ induction.

An effective vaccine should induce both humoral and cellular immuneresponses that contribute to protection (Zinkernagel (2003) Annu. Rev.Immunol. 21, 515-546). Humoral immunity involves antibody production byB cells that act to neutralize an extracellular pathogen, its proteins,and toxins, while cellular immunity involves production of cytokines andcytolytic capacities of T cells and acts to eradicate intracellularpathogens. Vaccines composed of either killed pathogen or purifiedproteins mixed with adjuvants act by priming humoral immunity (Meyer etal. (1974) J. Infect. Dis. 129 (Suppl.), S13-S18; Heath et al. (1998)Vaccine 16, 1131-1137). In contrast, live attenuated vaccines ofvirulent pathogens, act by priming cellular immunity (Levine & Sztein(2004) Nat. Immunol. 5, 460-464). The importance of cellular immunity inproviding vaccine protection against Y. pestis has been demonstratedusing a mouse model (Parent et al. (2005) Infect. Immun. 73, 7304-10).This report shows the importance of CD4 and CD8 T cells in immunity toY. pestis and that IFNγ and TNFα secreted by these cells played animportant role in it.

Early plague vaccine research centered on the bubonic form of disease.Heat-killed cultures of virulent Y. pestis formulated as vaccine wereused by Haffkine in 1897 (Haffkine (1897) Br. Med. J. 1, 1461). Kolle &Otto found that live attenuated Y. pestis strains protected mice againstvirulent infection (Kolle & Otto (1904) Z. F. Hyg. 48, 399-428). Thoughthese live attenuated strains were used in humans and their safety andefficacy was established (Strong (1908) J. Med. Res. 18, 325-346), theyoccasionally caused adverse reactions. Pneumonic plague vaccine effortshave largely focused on the development of subunit vaccines usingrecombinant Y. pestis proteins (Titball & Williamson (2004) Expert Opin.Biol. Ther. 4, 965-973); fraction 1 (F1) and V have been widely testedin vaccinations and these recombinant proteins protects mice againstpneumonic plague (Williamson et al. (1995) FEMS Immunol. Med. Microbiol.12, 223-230; Anderson et al. (1996) Infect. Immun. 64, 4580-4585;Andrews et al. (1996) Infect. Immun. 64, 2180-2187). A recombinant F1-Vfusion protein vaccine has been reported to protect mice (Heath et al.(1998) Vaccine 16, 1131-1137), but does not fully protect non-humanprimates against pneumonic plague.

The current treatment regimen for plague comprises use of theantibiotics tetracycline, streptomycin, and chloramphenicol. Recentlygentamicin, chloramphenicol, doxycycline and ciprofloxacin have alsobeen recommended.

Burkholderia models. Burkholderia mallei, is a category B biowarfareagent per the CDC classification. B. mallei is a Gram-negative,non-motile bacillus, and causes glanders primarily in horses, mules, anddonkeys. An embodiment of the present invention comprises a burkholderiadisease model. It infects by the oral route and spread is by closecontact with infected animals. Infection of horses is most oftenmanifested as a slow progressive, chronic disease, whereas in donkeys,the disease is usually severe, causing death in 7-10 days (Acha &Szyfres (1987) Zoonoses and communicable diseases common to man andanimals. 2nd ed. Washington, DC: World Health Organization). Otheranimals such as mice, hamsters, guinea pigs, monkeys, and dogs, are alsosusceptible to this pathogen (DeShazer 2004). B. mallei can also infectby the cutaneous route. There is no effective treatment for glanders inthe natural host, and animals diagnosed with glanders are typicallyisolated and destroyed. In humans, infection with B. mallei can occurvia mucosal (oral, nasal, ocular) or cutaneous routes. Currently, thereis no vaccine against B. mallei. Because of its potential use as abiowarfare agent, a vaccine against B. mallei is a high priority.Indeed, B. mallei was used as a bioweapon in the First World War byGerman troops to disable the Russian army's horses and mules (Aldhous(2005) Nature 434, 692-3).

A genetically related species, Burkholderia pseudomallei, causesmelioidosis which has a fatality rate of almost 50% in countries such asThailand (Aldhous (2005) Nature 434, 692-3). It is endemic in parts ofAsia and in northern Australia. B. pseudomallei is also considered apotiential bioterrorism agent. Another species, Burkholderiathailandensis, is considered non-pathogenic in humans.

The complete genome sequence of B. pseudomallei (Holden et al. (2004)Proc. Natl. Acad. Sci. USA 101, 14240-14245) has been determined by theWellcome Trust, UK, and that of B. mallei (Nierman et al. (2004) Proc.Natl. Acad. Sci. USA 101, 14246-51) was determined by TIGR. It appearsthat B. mallei evolved from B. pseudomallei by deletion of portions ofits genome (Godoy et al. (2003) J. Clin. Microbiol. 41, 2068-2079).There are reports that gene losses have contributed to the pathogenicevolution of bacterial species (Maurelli et al. (1998) Proc. Natl. Acad.Sci. USA 95, 3943-3948; Moore et al. (2004) Infect. Immun. 72, 4172-87).The non-pathogenic B. thailandensis has the ability to assimilatearabinose using proteins encoded by an arabinose assimilation operon,while the pathogenic species B. mallei and B. pseudomallei have lostthis operon. Thus, genes for arabinose assimilation have been termedanti-virulence genes (Moore et al. (2004) Infect. Immun. 72, 4172-87).

Analysis of the genome sequence of B. pseudomallei identified severalgenes encoding survival and virulence functions, including three typeIII secretion system (TTSS) genes, while in B. mallei genes responsiblefor the virulence function form a gene cluster encoding anexopolysaccharide capsule (DeShazer et al. (2001) Microb. Pathogen. 30,253-269) and a TTSS (Ulrich & DeShazer (2004) Infect. Immun. 72,1150-1154). The TTSS was found to be essential for intracellularsurvival of Burkholderia mallei within human macrophage-like cells(Ribot & Ulrich (2006) Infect. Immun. 74, 4349-53).

Several animal species have been used a models of human B. malleiinfection, including monkeys, guinea pigs, hamsters, and mice. Acuteglanders in humans is characterized by rapid onset of pneumonia; as aresult, an aerosol model of infection in Balb/c mice was developed(Lever et al. (2003) J. Med. Microbiol. 52, 1109-15). In the initialstage of the disease, the pathogen localizes in the upper and lowersections of the respiratory tract and is transported by alveolarmacrophages to regional lymph nodes. As disease progresses, bacteriadisseminate and are also found in other organs, including liver andspleen, and in the bloodstream in later stages.

In the host immune response to B. mallei infection, type I cytokines,IFNγ and IL-12, are key in controlling the initial infection (Rowland etal. (2006) Infect. Immun. 74, 5333-40). In that report, increased levelsof IFNγ, IL-6, MCP-1, IL-12p35, IL-18, and IL-27 were found in the serumand spleen of peritoneally-infected mice. IFNγ knockout mice were unableto control infection and died within 2-3 days, suggesting the importanceof IFNγ in host immunity to B. mallei infection. B. mallei has an outerlipopolysaccharide (LPS) capsule. It has been reported that B. malleiLPS is a potent activator of human Toll-like receptor (TLR) 4 (Brett etal. (2007) Mol. Microbiol. 63, 379-90), eliciting TLR4-mediatedstimulation of human macrophage-like cells (THP-1, U-937),monocyte-derived macrophages, and dendritic cells, resulting in highlevels of TNF-a, IL-6, and RANTES. These observations suggest that theB. mallei LPS capsule plays an important role in the pathogenesis of thehuman disease.

As B. mallei and B. pseudomallei are naturally soil-dwelling microbes,they are intrinsically resistant to many antibiotics. In a study on 65isolates of B. mallei and B. pseudomallei, a wide range of resistance toantimicrobial agents was noted, including to fluoroquinolones, β-lactamantibiotics, aminoglycosides, and macrolides. Bacteria were found to besusceptible to imipenem, ceftazidime, piperacillin, piperacillin /tazobactam, doxycycline, and minocycline (Thibault et al. (2004) J.Antimicrob. Chemother. 54, 1134-8). These antibiotics are currentlybeing used in post-exposure treatment of melioidosis and glanders.

A report indicated that pretreatment of Balb/c mice with anoligodeoxynucleotide (ODN) containing CpG motifs (CpG ODN 7909)protected them against aerosol challenge with B. mallei. This protectionwas found to be associated with enhanced levels of interferon gamma(IFNγ)-inducible protein 10 (IP-10), IL-12, IFNγ, and IL-6. Thus,treatment with CpG ODN 7909 provided an effective pre-exposure therapyto protect against glanders. CpG ODN 7909 is an agonist of TLR9. TLRs 7and 9 are thought to share the MyD88-dependent pathway that activatesinterleukin-1 receptor-associated kinases (IRAKs) and TRAF6 locateddownstream (Kawai & Akira (2005) Arthritis Res. Ther. 7, 12-19). These,in turn, activate NF-κB and mitogen-activated protein (MAP) kinases,leading to activation of inflammatory cytokine genes. Thus, TLR agonistsactivating TLR7/9 or other TLRs may act to protect against lethalinfection with glanders.

In summary, in the diseases described above, the pathogenesis of HIV,Yersinia, Burkholderia, filoviruses, and tularemia all involveantigen-presenting cells being infected with a pathogen, which they thentransport to regional lymph nodes where pathogen replication occurs. Asan example, the in vitro infectious disease model can be comprised oftwo modules: an infection module, where the macrophages/DCs are infectedby the pathogen, which can be represented in model systems of thepresent invention by the VS, and the disease module, which representsthe process whereby infected APCs transport the pathogen to regionallymph nodes, which can be represented in the model systems of thepresent invention by the LTE. In this disease model module LTE, thelymphocytes are activated/primed and the pathogen multiplies, as occursnaturally in a human lymph node. For this category of pathogen, the LTEitself may serve as the disease model module rather than needing aseparate engineered tissue construct as with, e.g., a tumor diseasemodel. Optionally, one could use the AIS to generate an immune responseto a potential vaccine candidate and then transfer the contents of thenon-infected, primed LTE to an infected LTE disease model module todetermine the effect of the vaccine candidate on viral replication andinfection, and the effect on immune clearance of the infection. Becausethe present invention is modular, with both infected and uninfected VSand LTE modules, and the modules can be used in different combinationsand sequences, a practioner may select different embodiments toinvestigate different effects on viral replication, infection, andpathogenesis resulting from different antigens, adjuvants, vaccines,drugs, and other agents, depending on whether the effect sought isprophylactic, therapeutic, or both, and depending on the desired site ofimmunity (e.g., peripheral tissue, lymphoid tissue, or both). Indeed,some embodiments of the invention may use only a modified VS or LTE asthe entire disease model for certain types of disease.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Schematic illustration of an embodiment of the invention,showing the integration of the AIS and a disease model.

FIG. 2. A 3D heterogeneous tissue construct, comprising the addition ofcells on the top and bottom of the construct, to create endothelial andepithelial layers.

FIG. 3. A schematic representation of the development of a genericdisease model and how it can be tested with a particular disease.

FIG. 4. Schematic illustration of, as an example, vertically expandingmelanoma tumor cells or bacterially or virally infected fibroblast cellsinside the 3D construct.

FIG. 5. Flow chart for preparation and analysis of an in vitro tularemiamodel.

FIG. 6. An example in vitro disease model architecture.

FIG. 7. HIV vaccine candidate testing flow chart.

FIG. 8. HIV disease model for testing HIV vaccine candidates. Acandidate vaccine is placed in the VS module to prime APCs. These APCsare then placed in the LTE module to prime lymphocytes. The invitro-immunized lymphocytes and resulting antibodies are placed in thedisease module along with infected APCs (obtained from a parallelinfection with HIV in the infection site module). Autologous cells areused. Any protection offered is quantitated, for example, by inhibitionof viral replication or cell lysis.

FIG. 9. Assessment of correlates of protection in an infectious disease.All elements of correlates of protection (CTL, antibodies, cytokines,CD4⁺ T cells) are examined.

FIG. 10. Diseased cells (e.g., HIV-infected APCs) would be exposed to‘output’ from the VS/LTE combination by putting the diseased cells(e.g., HIV-infected APCs) from the VS into the LTE to evaluate theeffect on the diseased cells. The effects on the LTE components by thediseased cells can be assessed, as can any effect on the diseased cellsto define the correlates of protection.

EXAMPLES

The artificial immune system of the present invention can be used tostudy many human diseases, including HIV. This in vitro model can notonly be used to model bacterial and viral disease systems, but also usedto study host immune responses in injury and inflammation.

Example 1

Generic tissue construct for a 3D in vitro disease model. FIG. 2illustrates a 3D heterogeneous tissue construct, comprising the additionof cells on the top and bottom of the construct, to create endothelialand epithelial layers. This model is an improvement on our established3D endothelial cell-only construct, which has been used fortransendothelial migration and for monocyte to dendritic cell andmacrophage differentiation (the vaccination site, VS).

The 3D model of this example can be used to study immunophysiologicalreactions when subjected to various diseases and vaccine formulations.This is a generic construct because most tissues involve a 3Dextracellular matrix with associated endothelial and epithelial layers.The disease, whether viral, bacterial, or tumoral, is introduced intothe generic tissue construct. The various immunocytes and biomoleculesfrom the AIS (e.g., antibodies, T cells, cytokines, chemokines) can thenbe delivered to the disease model to examine and detect effectorresponses (e.g., the presence or absence of neutralizing antibodies,cytotoxicity).

Example 2

Tumor modeling in the AIS using melanoma cells. Many in vitro modelsystems have been used for examining the effects of anti-cancertherapeutics and tumor growth in adult and childhood cancers, using bothprimary cells and various cell lines (see, e.g., Houghton et al. (2002)Clin Cancer Res 8, 3646-57). Such models have proven useful forassessing tumor metabolic states, inhibition of proliferation, anddecreases in overall biomass (see, e.g., Monks et al., (1991) J NatlCancer Inst 83, 757-66; Scherf et al., (2000) Nat Genet 24, 236-44).

Animal models of human cancers have not been good predictors of humantherapeutic outcome because of species differences (see, e.g., Houghtonet al.,(2002) Clin Cancer Res 8, 3646-57; Bridgeman et al.,(2002) CancerRes 60, 6573-6; Batova et al.,(1999), Cancer Res 59, 1492-7).

As with any tumor model, the primary end goal is to increase patientsurvival and overall well being and to decrease tumor burden. The mostpredictive model will aid in correlating between what is observed invitro with what is observed in the clinical setting.

Melanocytes in human skin are inter-follicular melanin-containing(pigmented) cells within the epithelial stratum and are ofneuroectodermal origin. Melanoma is a common form of human skin cancer.Malignant melanoma (both pigmented and non-pigmented forms) arefrequently resistant to interventional therapies and are associated withsignificant morbidity and mortality.

Two modes of melanoma cellular proliferation are known to occur: one ina radial direction and the other in a vertical direction, into thesubepithelial matrix (dermal layer in vivo) (Chudnovsky et al., (2005)Nat Genet 37, 745-9). Many factors have been implicated in spontaneous,uncontrolled proliferation including genetic alterations, overexpressionof the catalytic subunit of human telomerase reverse transcriptase(TERT) and expression of melanoma markers HMB-45 and Melan-A. Pagetoidinvasion into upper epithelial and dermis layers is typically observedunder these conditions. Various melanoma cells can be purchased fromATCC (e.g., A-375, SK-Mel-31, WM115, SK-Mel-2, SK-Mel-24) with varyingcharacteristics as to invasion properties (vertical or radial) andexpression of specific human melanoma markers (e.g., NRAS, PI3K, CDK4,HMB-45 or Melan-A).

Example 3

Heterogeneous tissue constructs with the addition of cells on the topand bottom of the tissue construct to form endothelial and epitheliallayers. A schematic representation of the development of the genericdisease model and how it can be tested with a particular disease isshown in FIG. 3. As an example, we used a polycarbonate membrane supportstructure to prepare a 3D ECM matrix, comprising either collagen,synthetic or natural materials (e.g., hydrogels, PLA, PLGA, gelatin,hyaluronic acid), or combinations thereof. We have established an ECMthat is capable of supporting two cell layers. We first grow a layer ofepithelial cells (e.g., human keratinocytes) on one side of the matrix.An advantage of this model is that other epithelial cells can be used,such as respiratory epithelial cells, skin epithelial cells, orintestinal/oral epithelial cells (as schematically illustrated in FIG.3). The basement membrane zone between the epithelium and the matrix isimportant to the success of this aspect of the construct and additions,such as collagen types IV or VII can be included. For a melanoma modelthe barrier function of the basement membrane may also be important indissecting the pathology of modes of metastasis. This is an advantage ofthe general architecture of the disease model of the present invention;it can be used to mimic many tissues by using different epithelial celltypes. After melanocyte and keratinocyte seeding and when thekeratinocytes have become established and begun stratification, thecells are exposed to an air interface, to encourage continuedstratification, formation of tight cell junctions, and keratinization.

When a keratinized cell layer is formed, the construct can be inverted,so that a layer of endothelial cells (e.g., HUVECs, immortalizedendothelial cell lines) can be applied to the other side. When theendothelial cells have established, the construct can be inverted (so itwill be upright again) to reinstate the air interface for thekeratinocytes. When the endothelial cells form a confluent monolayer,the tissue construct is complete and ready for characterization.

In other embodiments of the present invention, in a multifunctionaldisease model without melanocytes in epithelial layer, a viral orbacterial disease model can be prepared. In these embodiments, eitherthe viral or bacterial component is applied to the specialized,non-keratinized epithelial surface, mimicking normal physiologic events.In viral and bacterial invasion/infection, epithelial compromise iscaused by either cellular infection or release of bacterial toxins,which can also be monitored.

Example 4

Viability of the 3D generic disease tissue constructs. Studies ofkeratinocytes have shown the cells to remain viable in culture forseveral weeks (Boelsma et al., (2000) Acta Derm Venereol 80, 82-8). Wealso have experience of maintaining HUVECs in culture and on a 3Dconstruct for several weeks. Viability of the cells on the construct canbe monitored by, for example, such methods as identifying anymorphological changes and by the classic LDH release assay. As cellsdie, the plasma membrane becomes leaky with LDH being released into theculture medium and can be measured with a coupled enzymatic assay thatresults in the conversion of resazurin into a fluorescent resorufinproduct. The amount of fluorescence produced is proportional to thenumber of lysed cells. Cell staining can also be performed on the tissueconstructs to measure live/dead cell populations. Cell-permeant esterasesubstrates, such as CellTracker Green CMFDA, serve as viability probesthat measure both cell membrane integrity, required for intracellularretention of the probe, and enzymatic activity, required to activate thefluorescence of the probe. Cell-impermeant nucleic acid stains, such asethidium homodimer-1, can be used to detect dead cells. Fluorescentlystained cells can then be observed by confocal microscopy.

Example 5

Epithelial cells form stratified layers on the constructs. For theconstruction of the skin equivalent model, the keratinocyte layer isexposed to an air interface to encourage formation of stratified layers.The formation of the stratified layers can be monitored by microscopicexamination. Periodically cell layers can be examined by usingimmunofluorescence confocal microscopy to identify the tight junctionsand nuclei of the cells. Additionally, samples can be fixed inparaformaldehyde, embedded in parafin, cut into sections, and stainedwith haematoxylin and eosin for light microscopic examination.

Example 6

Construction of a generic tissue module creating an in vitro diseasemodel. In embodiments of the present invention, the 3D model is examinedto observe immune- or inflammation-mediated responses to variousdiseases (e.g., tumors models). As examples, melanoma cells, HSV,influenza virus, Escherichia coli, and Staphylococcus aureus are used.

Melanoma cells are incorporated when the epithelial layer is formed. Ashuman melanocytes are interfollicular, basal epithelial cells, using acell line that is slower growing allows keratinized epithelialformation. Application of different cell types can be accomplished byintermixing these cells with normal keratinocytes (for example, at aratio of ˜5×10³ to ˜30×10³). Monitoring of the vertical and lateralspread of the malignant melanocytes can be accomplished by staining withfluorochrome-labeled, melanocyte-specific markers and confocalmicroscopy. As another example, other constructs can be digested and thenumber of melanocytes present can be assessed using flow cytometry andsimilar markers.

Example 7

As an example, a methodology that can be used to add verticallyexpanding melanoma tumor cells or bacterially or virally infectedfibroblast cells inside the 3D construct, is illustrated schematicallyin FIG. 4. To add tumor cells to the disease model, we mix these cellswithin the ECM material before it is added to the membrane support andbefore we begin to grow the epithelial and endothelial cells on thematrix.

Example 8

For the preparation of a viral model, there are several relevantmethods. As an example, for a live virus, we would infect an epitheliallayer. As another example, virus-infected irradiated fibroblasts can beincorporated in the collagen matrix. HLA-matched, syngeneic orautologous fibroblasts can be used; they can be propagated and infectedwith virus at an appropriate multiplicity of infection (MOI) (e.g.,˜10). Infection is allowed to proceed until an appropriate timepost-infection, at which time infectious virus is UV-inactivated.

Example 9

In vitro infection/disease models are important for an analysis of theviral life cycle, including attachment, entry, and uncoating, and tounravel the interactions between viral particles and host target cells.We can also use the in vitro disease/infection model to examine theefficacy of the vaccine-induced immune products created in the AIS.Suitable example viral disease models include Herpes simplex viruses(HSV) and influenza viruses. Human and/or murine model systems can beused.

Example 10

The present invention comprises both two- and three-dimensional (2D, 3D)models of infection/immune induction. In an example 2D model, a staticculture system can be employed. In an example 3D model, the vaccinationsite (VS) and lymphoid tissue equivalent (LTE) can be used.

Example 11

Several methods of viral antigen introduction are suitable forpracticing the present invention. As an example, direct infection ofcultured epithelium with virus at an appropriate multiplicity ofinfection (MOI) can be used. As another, example, HLA-matched orsyngeneic fibroblasts can be used; they can be propagated and infectedwith virus at an appropriate MOI (e.g., ˜10). Infection will be allowedto proceed until an appropriate time post-infection at which timeinfectious virus will be UV-inactivated. The kinetics of virus infectionand inactivation can be confirmed by, for example, immunofluorescenceand plaque assay, respectively.

Infectious virus or virus-infected UV-inactivated fibroblasts can beadded to the cultures. For fibroblast cultures, uninfected UV-treatedfibroblasts can be used as negative controls. In 2D cultures, infectiousvirus, fibroblasts or vaccine/adjuvant formulations are added to a mixedimmunocyte population containing antigen presenting cells (APCs) andlymphocytes. For 3D culture, antigens are introduced into a vaccinationsite (VS) containing reverse-transmigrated (RT) antigen presenting cells(APCs), comprising dendritic cells (DCs). APCs then process the antigenand are introduced into the lymphoid tissue equivalent (LTE), comprisingT and B lymphocytes.

In both 2D and 3D cultures, immunological parameters of interest includepatterns of immunocyte phenotype and cytokine synthesis and secretion.Flow cytometric analysis is valuable in this regard. Virus-specificcytotoxic activity can be assessed for T cells using, for example, anon-radioactive LDH cytoxicity assay with virus-pulsed target cells. Bcells can be evaluated for specificity and isotype of antibodysecretion, as well as neutralizing capability.

To evaluate recall responses and anti-viral activity, immunocytes and/orsoluble factors can be recovered from 2D cultures or from the LTE of the3D system for analysis. These immunocytes and/or biomolecules can thenbe tested, for example, using an in vitro 2D, an in vitro 3D tissueengineered disease model, or an in vivo (especially murine) diseasemodel. In 2D experiment, these can be co-cultured with, for example,suspension or monolayer cultures of fibroblasts. The cultures can thenbe challenged with infectious virus or virus-infected UV-inactivatedcells. As another example, a similar in vitro challenge can be performedin the 3D tissue engineered disease model incorporating an epitheliallayer.

In the in vitro experiments, cultures are harvested at selected timespost-challenge and assayed for virus-specific immunity and anti-viralactivities, as indicated, for example, by titers of infectious virusrecovered.

To assess the in vivo efficacy of immunocytes derived from the LTE, wecan conduct adoptive transfer studies in, for example, a mouse modelwhere selected cell populations derived from the AIS can be introducedprior to viral challenge. Several murine models of HSV infection areavailable and can be used to assess protective efficacy of cellsrecovered from the AIS.

Example 12

As another example of the present invention, we can conduct an‘experiment of nature’ involving seropositive individuals with recurrentHSV (S⁺R⁺), seropositive individuals without recurrent disease (S⁺R⁻)and seronegative (S⁻R⁻) human subjects. Cells from these subjects can besensitized with viral antigens. Subsequent immunological read-outs canallow for discrimination of primary and recall immune events and immuneprofiling of protective immune mechanisms when comparing S⁺R⁺ and S⁺R⁻subjects.

Example 13

In a melanoma tumor model, the spread of the melanocytes radiallythrough the epithelial layer and penetration into the sub-epithelialmatrix (vertical tumor expansion) can be examined. As some melanoma celllines exhibit radial expansion only (possibly the result of theimpediment of the basement membrane structure or biochemical makeup ofthe different collagens) or vertical expansion only, it is possible totarget the immunocyte population within the matrix. The presence ofmelanoma antigen with or without the addition of adjuvants, will lead tothe maturation of DCs that have captured antigen.

As the APCs reverse transmigrate out of the module with capturedantigens, they can be matured with TNFα. APC phenotypic markers and apanel of inflammatory cytokines can be compared to modules withoutmelanoma cell additions. These results can then be compared to VSresponses with known stimulants or adjuvants (such as LPS, CpG,poly(IC), MF59). Functional assessment of these monocyte-derived APCsafter exposure to tumor antigens from the melanoma cells in the VS, canbe conducted by placement into the LTE module for assessment of antigenpresentation. IL12 is an important cytokine released by DCs activating Thelper cells, which then release IFNγ. IFNγ contributes to CTL activityand B cell differentiation into plasma cells. Antibody release,compliment fixation, and influx of PMNs to the region of the tumor cells(in vivo) causes release of TNFα. TNFα and IFNγ have tumor cytostaticproperties. (Croci et al. (2004) Cancer Res 64, 8428-34) and can bemonitored. As an example, a non-radioactive cytotoxicity T cell assaymonitoring LDH release can be used.

Example 14

Tularemia model. In an embodiment of the invention, an in vitro model oftularemia is prepared. Live attenuated strains can be safely used in aBSL-2 laboratory. PBMCs from a series of human blood donors are preparedand mixed with different ratios of F. tularensis in a collagenous 3Dmatrix. The immune response to F. tularensis is then assessed, using,for example, the Bioplex 22-cytokine kit. For example, levels of IL-18,IL-1β, IFN-γ, IL-12, IL-4, and IL-5 are determined. IgM and IgG antibodyELISAs are conducted on culture supernatants to examine the humoralresponse. The collagenous construct is digested with collagenase torelease the cells, and aliquots are serially diluted and plated onchocolate-agar plates. Colonies are counted after ˜3 days to determinethe multiplication of pathogen in host cells. The activation state of Tcells, B cells, and macrophages in response to infection is determinedby, for example, FACS analysis. A flowsheet of the experiment is shownin FIG. 5.

To determine whether protection is offered by secreted antibodies, theculture supernatants from first infection are added to a new set ofPBMCs and F. tularensis in collagen to permit a new infection. This isfollowed by determining cytokine levels, ELISA, flow cytometricanalysis, and colony counts, as described above. Any protection offeredby antibodies is expected to be reflected in increased protective immuneresponses and lowering of pathogen colony counts.

This in vitro model can be used as test bed for vaccine and drugcandidates. Quinolone drugs, such as ciprofloxacin, have been reportedto be effective against tularemia (Johansson et al. (2002) Scand. J.Infect. Dis. 34, 327-30). The effect of such drugs can readily be testedby the addition of the drug to the in vitro model and determining anyreduction in colony counts. Similarly, other drugs and vaccinecandidates could be assessed for efficacy in this model system.

Example 15

Filovirus models. The vaccination site (VS) module of the artificialimmune system of the present invention, comprising a confluentendothelium with monocytes and macrophages, can be used as apathogenesis model system. For the disease module, the VS and InfectionSite (IS) modules can be used interchangeably, unless otherwise noted.To prepare a filovirus disease model, as an example intracellular viralpathogen, IS modules are prepared and serve as infection targets.Preparation of the IS module comprises growing a monolayer ofendothelial cells over a collagen matrix. PBMCs are added on top of theendothelial cell layer. Monocytes preferentially extravasate across theendothelium into the collagen matrix, with some T and B cells (5-10%).Monocytes in PBMCs differentiate into APCs of a range of phenotypes asthey traverse the endothelium. Some monocytes differentiate into matureand immature dendritic cells (DCs) and then reverse transmigrate acrossthe endothelium from the collagen, while other monocytes differentiateinto macrophages and these sub-endothelial cells remain in the collagenmatrix. The system models the in vivo differentiation process, wherebyAPCs (e.g., monocytes) entering a site of vaccination obtaindifferentiation signals as they cross the endothelium into the tissue.This method is superior to the widely used cytokine-induced (e.g.,GM-CSF, IL-4, MCSF) methods of generating DCs and macrophages fromPBMCs.

Infectious virions can be added on top of the endothelial cells.Filoviruses will infect the endothelium and also migrate across it toinfect DCs and macrophages in the collagen. Additional lymphocytes canthen be included in the collagen to mount an immune response against thepathogen.

Thus, the VS module/infection site (IS) module is the essential elementof the Ebola disease model, the only difference is that we willincorporate additional lymphocytes. The system can be incubated fordifferent time periods to study progression of disease. Althoughendothelial cells are an important part of the IS system, the presenceof endothelial cells in the system may generate MHC-I-mediatedallogeneic responses in response to Ebola virus or VSV expressing Ebolaviral proteins or the vaccine candidates. If they do, APCs are isolatedfrom the IS module after 48 h (when DCs and macrophages havedifferentiated in the IS) and add them with fresh PBMCs in the LTEmodule/disease module for stimulation. If the endothelial cells do notgenerate allogeneic responses, we will continue to use the IS as thedisease module.

Thus, the VS/IS module of the artificial immune system of the presentinvention closely mimics the in vivo scenario of infection; all of thecomponents involved in the infection process, including endothelialcells, DCs, macrophages and lymphocytes, are present in the modelsystem. The system can be incubated for different time periods to studyprogression of disease. Cytokine profiles are assessed using, forexample, a Bioplex assay and antibody responses are assessed by, forexample, ELISA at different time points using culture supernatants fromthis model. For example, levels of IFNα, IL-6, MCP-1, MIP-1a, MIP-1b,IFN-β, IFN-γ, IL-18, and TNF-α are determined. Disease-associatedfactors, such as TF, can also be measured. The collagen matrix isdigested with collagenase to release the cells; their apoptotic stateand expression levels of different cellular markers on endothelialcells, DCs, macrophages, and lymphocytes are assessed by, for example,flow cytometry. Antibodies generated can also be assessed.

To assess the efficacy of filovirus vaccines, PBMC cells from a seriesof human blood donors are incubated with different concentrations (e.g.,˜1-50 μg/mL) of, for example, Ebola vaccine in the VS module and in asimple collagenous 3D matrix and are incubated for different timeperiods. The vaccine formulation will be taken up by monocytes, in asimilar way to infectious virions, resulting in the establishment of ahost immune response to the vaccine antigens. This host immune responseto the test vaccine is then assessed in terms of, for example, cytokineresponses, using, for example, the Bioplex 22-cytokine kit. For example,levels of IFNα, IL-6, MCP-1, MIP-1a, MIP-1b, IFNβ, IFNγ, IL-18, and TNFαare determined. IgM and IgG antibody ELISAs are conducted with culturesupernatants to determine the levels of antibody production in responseto the test vaccine. The collagenous construct is digested withcollagenase to release the cells and the activation and apoptotic stateof T cells, B cells and monocytes, macrophages, and dendritic cells inresponse to infection are determined by FACS analysis. Experiments usingvectors expressing different viral proteins, peptides, and combinationsof proteins can also be used to assess differences and variations in thehost immune responses to viral proteins.

Example 16

Yersinia model. An embodiment of the present invention comprises adisease model of Yersinia pestis infection. In an infection module(e.g., the IS), macrophages are infected by the pathogen. The diseasemodule models the process by which infected macrophages transport thepathogen to the regional lymph nodes. In this disease module, thelymphocytes are activated/primed and the pathogen multiplies. This lymphnode equivalent (LTE) module serves as the disease module. In vivo, thebacteria replicate in lymph nodes before migrating to other organs. Theartificial immune system of the present invention is flexible in thatthe various modules are designed as plug-and-play immunologicalconstructs. This feature is exploited here with infection and diseasemodules of the Yersinia infectious disease model.

The infection module system (IS) involves growing a monolayer ofendothelial cells over a collagen matrix. PBMCs are added on top of theendothelial cell layer. Monocytes preferentially extravasate across theendothelium into the collagen matix with a small number of T and B celllymphocytes (5-10%). The monocytes in the PBMCs differentiate into APCsof a range of phenotypes as they traverse the endothelium. Somemonocytes differentiate into mature and immature dendritic cells (DCs)and reverse transmigrate across the endothelium from the collagen, whileother monocytes differentiate into macrophages and these sub-endothelialcells remain in the collagen matrix. This system mimics the in vivodifferentiation process, where antigen-presenting cells (e.g.,monocytes) entering the site of vaccination receive differentiationsignals as they cross the endothelium into the tissue.

To prepare the disease model, Y. pestis is added at differentmultiplicities of infection (MOI) on top of the endothelial cells of theIS module. Y. pestis crosses the endothelium and enters the collagenwhere the pathogen will infect the macrophages and also pulse thedendritic cells with Y. pestis antigens. After 4-6 h of infection, thecollageneous matrix is digested to release the APCs.

These infected APCs from the IS are then transferred to the diseasemodule (LTE) where they are added with autologous PBMCs and can beco-cast in collagen. The lymphocytes in collagen mount an immuneresponse against the pathogen. The system can be incubated for differenttime periods to study disease progression. The disease manifestation isobserved in terms of, for example, the development of severe necroticinflammation, macrophages and dendritic cells undergoing apoptosis as aresult of infection and inflammation, and the host immune systemmounting an immune response to contain the infection. Containment ofinfection will be reflected as reduction in bacterial counts. Hostmacrophages, DCs, and lymphocytes will secrete pro-inflammatorycytokines and chemokines and antibodies in response to the infection.

Parameters of disease pathogenesis will be determined as follows.Collagen constructs from the disease module are paraffin-embeddedfollowed by sectioning and microscopy to examine the inflammatorylesions. Collagen is digested with collagenase to release cells, whichare then assayed for apoptotic and other cellular expression markers byflow cytometry. This will provide information about host cell death andthe activation profile of the immune cells due to the infection. Analiquot of the collagenase digest will be plated on nutrient agar platesto determine the colony forming units (cfu) and hence survival andmultiplication of pathogen in the host cells and containment of theinfection. Culture supernatants are assayed for cytokine and antibodysecretion. The cytokine profiles are determined by, for example, aBioplex assay and antibody responses are assessed, for example, by ELISAat different time points. For example, levels of IL-6, MCP-1, IL-12p35,IFNγ and TNFα can be determined. IgM and IgG antibody ELISAs areconducted using culture supernatants to determine the levels of antibodyproduction in response to the infection.

In further embodiment of the present invention, the infectious diseasemodel is used to test potential therapeutic methods that may cure theinfection, pre- and/or post-exposure. The model can also be used toaddress basic questions related to bacterial pathogenesis.

The effects of various TLR agonists and vaccine adjuvants have beenexamined using the VS system. It has been reported that human monocytesinfected with Y. pestis express cell surface TLR9 and differentiate intodendritic cells (Shaikh 2004). The effects of CpG and/or other adjuvantsand Y. pestis infection in modulating DC function as APCs and on abilityto contain the infection, can readily be tested in the model system ofthe present invention by pretreating the infection module with TLRagonists for 24-48 h, followed by introduction of the pathogen for 4-6h. Infected APCs are taken out of the infection module and are thenadded to the disease module for different periods. The collagenousmatrix from the disease module is digested with collagenase. Theprotection offered by, for example, adjuvants can be quantitated byplating the collagenase digest (as described above) and estimating thereduction in cfu.

Cytokines play important roles in the control of infection. ExtraneousIFNγ and/or TNFα or IL-12 can be added in different concentrations tothe disease module together with APCs from the infection module. Thecontrol of infection can be studied, for example, in terms of reductionin cfu by, for example, plating method and by, for example, determiningcytokine and antibody levels in culture supernatants.

The TTSS and Yops are important for Y. pestis pathogenesis, as discussedearlier. Their effects can be tested in the infection module by addingthese antigens to it and transferring the affected APCs to the diseasemodule and determining the ability to contain the infection, by, forexample, measuring the reduction in cfu by, for example, a platingmethod.

Monoclonal antibodies have been produced against Y. pestis and have beenreported to protect Balb/c mice against Y. pestis challenge (Eyles etal. (2007) Vaccine 25, 7301-6). The protective effect of thesemonoclonals can be tested in the disease module system by addingprotective or therapeutic antibodies to it. The infected APCs from theinfection module, together with protective antibodies and autologousPBMCs, can then be co-cast in the disease module for different periods.Protection by the antibodies is determined, for example, by reduction incfu by, for example, a plating method. Additionally, antibodies in theculture supernatants in the disease module, produced in response to theinfection, can be added to a new infection set and any protectionoffered can be determined, for example, by cfu reduction, using, forexample, a plating method.

In another embodiment of the present invention, vaccine candidates, suchas those using the F1 or V antigen, can be tested for efficacy byplacing in vitro-immunized lymphocytes in the disease module. First, thevaccine is placed in the VS module to prime APCs. These APCs are thenplaced in the LTE module to prime lymphocytes, which will be used in thedisease module. These in vitro-immunized lymphocytes are placed in thedisease module along with infected APCs (obtained from a parallelinfection with Y. pestis in another infection module). Autologous PBMCswill be used throughout. The protection offered can then be quantitated,by for example, reduction in bacterial cfu by, for example, a platingmethod.

Example 17

Burkholderia model. An embodiment of the present invention comprises anin vitro disease model of Burkholderia mallei using the artificialimmune system, based on multidimensional interrogation of humanleukocytes. The artificial immune system can rapidly provide informationabout the effects of an immunotherapy on human population subgroups(genetic diversity, HLA haplotypes, age, gender).

In the infection module (IS) of the artficial immune system of thepresent invention, macrophages are infected by the pathogen (the ISmodule). The disease module models the process whereby infectedmacrophages transport the pathogen to the regional lymph nodes. In thisdisease module, lymphocytes are activated/primed and the pathogenmultiplies. This LTE module is the disease module of the in vitrosystem. The bacteria replicate in lymph nodes before migrating to otherorgans. The artificial immune system is flexible in that the variousmodules are ‘plug-and-play; immunological constructs. This feature isexploited in an embodiment of the present invention, comprising theinfection and disease modules of a Burkholderia infectious diseasemodel.

Preparation of the infection site module involves growing a monolayer ofendothelial cells over a collagen matrix. PBMCs are added on top of theendothelial cell layer. Monocytes preferentially extravasate across theendothelium into the collagen matrix with a small number of T and Bcells (˜5-10%). The monocytes in the PBMCs differentiate into APCs of arange of phenotypes as they traverse the endothelium. Some monocytesdifferentiate into mature and immature dendritic cells (DCs) and reversetransmigrate across the endothelium from the collagen, while othermonocytes differentiate into macrophages and these sub-endothelial cellsremain in the collagen matrix. The system mimics the in vivodifferentiation process whereby antigen-presenting cells (e.g.,monocytes) entering the site of vaccination receive differentiationsignals as they cross the endothelium into the tissue.

In the IS, cells are infected by adding B. mallei at differentmultiplicities of infection (MOI) on top of the endothelial cells. B.mallei crosses the endothelium and enters the collagen where thepathogen will infect the macrophages and also pulse the dendritic cellswith B. mallei antigens. After 4-6 h of infection, the collageneousmatrix will be digested to release the APCs.

These infected APCs from the IS will then be transferred to the diseasemodule where they are added with autologous PBMCs and can be co-cast incollagen. The lymphocytes in collagen mount an immune response againstthe pathogen. The system can be incubated for different time periods tostudy disease progression. The disease manifestation is observed interms of measurable events or parameters, such as development ofnecrotizing lesions, macrophage and lymphocyte death, by apoptosis, as aresult of infection and inflammation, and the host immune response tocontain the infection. Containment of infection will be reflected as areduction in bacterial counts. Host macrophages, DCs, and lymphocyteswill secrete proinflammatory cytokines and chemokines and alsoantibodies in response to the infection; these can also be assessed.

Parameters of disease pathogenesis will be determined. The collagenconstructs from the disease module will be paraffin-embedded, followedby sectioning and microscopy to visualize necrotic lesions. Collagenwill be digested with collagenase to release cells, which will be thenassayed for apoptotic and other cellular expression markers by flowcytometry. This will provide information on host cell death and theactivation profile of the immune cells due to the infection. An aliquotof the collagenase digest will be plated on Mueller-Hinton agar platesto determine the colony forming units (cfu) and hence survival andmultiplication of the pathogen in the host cells, and the containment ofthe infection. Culture supernatants will be assayed for cytokine andantibody secretion. The cytokine profiles can be determined, forexample, by a Bioplex assay and antibody responses can be examined, forexample, by ELISA at different time points. For example, levels of IL-6,MCP-1, IL-12p35, IFNγ, IL-18, IP-10, and TNFα can be determined. IgM andIgG antibody ELISA can be conducted using culture supernatants todetermine the levels of antibody production in response to theinfection.

In another embodiment of the present invention, the infectious diseasemodel can be used to test potential therapies that may cure theinfection pre- and/or post-exposure. The model may also be used toaddress basic questions related to bacterial pathogenesis.

Studies with different types of TLR agonists and vaccine adjuvants havebeen conducted using the VS system. The protective effects of CpG and/orother adjuvants can readily be tested by pretreating the infectionmodule with TLR agonists for 24-48 h, followed by introduction of thepathogen for 4-6 h. Infected APCs are taken out of the infection moduleand are then added the disease module for different periods. Thecollagenous matrix from disease module can be digested with collagenase.The protection offered by the adjuvants will be quantitated by platingthe collagenase digest (as described above) and estimating the reductionin cfu.

As type I cytokines are important in control of initial infection(described above), extraneous IFNγ and/or IL-12 will be added indifferent concentrations to the disease module together with APCs frominfection module. The control of infection will be studied in terms ofreduction in cfu, by, for example, a plating method, and also thecytokine and antibody levels determined in culture supernatants.

The animal-type TTSS is important for B. mallei pathogenesis. This canbe tested in the infection and disease module by using RD01 and RD02mutant strains (Ribot & Ulrich (2006) Infect. Immun. 74, 4349-53).

Monoclonal antibodies have been produced againt B. mallei and reportedto passively protect Balb/c mice against B. mallei aerosol challenge(Treviño et al. (2006) Infect. Immun. 74, 1958-61). The protectiveeffect of these monoclonals can be tested in the disease module systemby adding protective or therapeutic antibodies to it. Infected APCs fromthe infection module together with protective antibodies and autologousPBMCs can be co-cast in the disease module for different periods.Protection by the antibodies is determined by reduction in cfu, by, forexample, a plating method. Additionally, the antibodies in the culturesupernatants in the disease module, produced in response to theinfection, can be added to a new infection set and any protectionoffered can be determined in terms of reduction in cfu, by, for example,a plating method.

Vaccine candidates can be tested in the disease model by placing invitro-immunized lymphocytes in the disease module. First, the vaccine isplaced in the VS module to prime APCs. These APCs are then placed in theLTE module to prime lymphocytes, which are then used in the diseasemodule. These in vitro-immunized lymphocytes are placed in the diseasemodule along with infected APCs (obtained from a parallel infection withB. mallei in the IS module). Autologous PBMCs are used throughout. Theprotection offered is quantitated by reduction in bacterial cfu, by, forexample, a plating method.

In another embodiment of the present invention, the disease model can beused to test new drug, vaccine, or therapeutic candidates for theirefficacy against glanders or melioidosis, diseases caused byBurkholderia mallei and B. pseudomallei.

Example 18

HIV models. HIV (human immunodeficiency virus) is the virus that causesAIDS (acquired immune deficiency syndrome). In vivo, HIV-1 infects DCsand they move to the lymph nodes where the virus infects CD4⁺ T cells.The lymph node then becomes the principal site of virus production. Inembodiments of the present invention, the steps of this process aremodeled by the modules of the artificial immune system.

In an embodiment of the present invention, HIV vaccine candidates aretested. As an example, HIV gp120 or gp140 vaccines can be assessed. HIVgp120/gp140-specific responses are assessed using PBMCs from human blooddonors. First, the candidate vaccine is placed in the VS module to primeAPCs. These primed APCs are then placed in the LTE module to primelymphocytes. The in vitro-immunized lymphocytes and antibodies generatedin the LTE are placed in the disease module along with infected APCs(obtained from a parallel infection with HIV in the infection sitemodule). Autologous PBMCs are used throughout. Any protection offered isquantitated by, for example, assessing inhibition of viral replicationor cell lysis.

Preparation of the infection site (IS) module involves growing amonolayer of endothelial cells over a collagen matrix. PBMCs are addedon top of the endothelial cell layer. Monocytes preferentiallyextravasate across the endothelium into the collagen matix with a smallnumber of T and B cells (˜5-10%). The monocytes in the PBMCsdifferentiate into APCs of a range of phenotypes as they traverse theendothelium. Some monocytes differentiate into mature and immaturedendritic cells (DCs) and reverse transmigrate across the endotheliumfrom the collagen, while other monocytes differentiate into macrophagesand these sub-endothelial cells remain in the collagen matrix. Thesystem mimics the in vivo differentiation process wherebyantigen-presenting cells (e.g., monocytes) entering the site ofinfection receive differentiation signals as they cross the endotheliuminto the tissue. This method is superior to the widely usedcytokine-induced (e.g., GM-CSF, IL-4, MCSF) methods of generating DCsand macrophages from PBMCs.

In an embodiment of the present invention, infectious virions can beadded on top of the endothelial cells. HIV will infect theantigen-presenting cells. Thus, the IS module of the artificial immunesystem of the present invention closely mimics the in vivo scenario ofinfection; all of the components involved in the infection process,including endothelial cells, DCs, macrophages and lymphocytes, arepresent in the model system. Infected APCs can then be transferred tothe LTE module.

The system can be incubated for different time periods to studyprogression of disease. Cytokine profiles can be assessed using aBioplex assay and antibody responses can be assessed by ELISA atdifferent time points using culture supernatants. For example, levels ofIFNa, IFN-γ, IL-18, IL-6, MCP-1, MIP-1a, MIP-1b, IFN-β, and TNF-α willbe determined.

To assess the efficacy of HIV vaccines, PBMC cells from a series ofhuman blood donors are incubated with different concentrations (e.g.,˜1-50 μg/mL) of HIV vaccine in the VS module and in a simple collagenous3D matrix and are incubated for different time periods. The vaccineformulation will be taken up by monocytes, in a similar way toinfectious virions, resulting in the establishment of a host immuneresponse to the vaccine antigens. This host immune response to the testvaccine is then assessed in terms of, for example, cytokine responses,using for example, the Bioplex 22-cytokine kit. For examples, levels ofIL-6, MCP-1, MIP-1a, MIP-1b, IFNβ, IFNγ, IL-18, and TNFα are determined.IgM and IgG antibody ELISAs are conducted with culture supernatants todetermine the levels of antibody production in response to the testvaccine. If the LTE comprises a collagen matrix, the collagenousconstruct is digested with collagenase to release the cells and theactivation and apoptotic state of T cells, B cells, and monocytes,macrophages, and dendritic cells in response to infection are determinedby FACS analysis. Experiments using vectors expressing different viralproteins, peptides, and combinations of proteins can also be used toassess differences and variations in the host immune responses to viralproteins.

In the IS, PBMCs are infected by adding HIV at different multiplicitiesof infection (MOI) on top of the endothelial cells. HIV infectsantigen-presenting cells in the IS. These infected APCs are thentransferred to the disease module/LTE module described above where theyare added with autologous PBMCs and can be co-cast in collagen. Thelymphocytes mount an immune response against the pathogen. The systemcan be incubated for different time periods to study diseaseprogression. The disease manifestation is observed in terms ofmeasurable events or parameters, such as development of macrophages andlymphocytes death and the host immune response to contain the infection.Containment of infection will be reflected as a reduction in viralcounts. Host macrophages, DCs, and lymphocytes will secreteproinflammatory cytokines and chemokines and also antibodies in responseto the infection; these can also be assayed in culture supernatants.

The cytokine profiles can be determined, for example, by a Bioplex assayand antibody responses can be examined, for example, by ELISA atdifferent time points. For example, levels of IL-6, MCP-1, IL-12p35,IFNγ, IL-18, IP-10, and TNFα can be determined. IgM and IgG antibodyELISA can be conducted using culture supernatants to determine thelevels of antibody production in response to the infection.

In an embodiment of the present invention, diseased cells (e.g.,HIV-infected APCs) would be exposed to ‘output’ from the VS/LTEcombination—either by transferring VS/LTE ‘output’ or by putting thediseased cells (e.g., HIV-infected APCs) into the LTE, in both cases, toevaluate the effect on the diseased cells. The effects on the LTEcomponents by the diseased cells can be assessed, as can any effect onthe diseased cells.

In an embodiment of the invention, IS-derived cells would be infectedbefore putting them into a naïve (uninfected) LTE, to assess the effectson the cells of the LTE. This models cells becoming infected in theperiphery before moving to the lymph node (or LTE). Effects on the LTEcomponents by the diseased cells can be assessed, as can effects on thediseased cells.

In another embodiment of the present invention, a naive (uninfected) LTEis infected to assess the effects on cells of the LTE.

While the foregoing specification teaches the principles of the presentinvention, with examples provided for the purpose of illustration, itwill be appreciated by one skilled in the art from reading thisdisclosure that various changes in form and detail can be made withoutdeparting from the true scope of the invention.

Any and all materials cited or referred to herein, including, but notlimited to, books, manuals, journal articles, abstracts, posters,websites, product literature, and other publications of any type ishereby expressed incorporated by reference in its entirety.

1. A method of evaluating a test agent in an in vitro diseased cellmodel, said method comprising: a) priming a culture of epithelial and/orendothelial cells with a test agent; b) adding a lymphoid tissue to theculture of a), thereby preparing a co-culture; c) transferring cells,culture media, or cells and culture media, from the co-culture of b) toa culture of diseased cells, wherein said diseased cells are immunesystem cells selected from the group consisting of PBMCs, monocytes,macrophages, dendritic cells, lymphocytes and antigen-presenting cells;and d) evaluating an effect of the test agent on the diseased cells inthe culture of c), thereby evaluating a test agent in an in vitrodiseased cell model.
 2. A method of evaluating a test agent in an invitro diseased cell model, said method comprising: a) co-culturingepithelial and/or endothelial cells with a lymphoid tissue in thepresence of a test agent; b) transferring cells, culture media, or cellsand culture media, from the co-culture of a) to a culture of diseasedcells, wherein said diseased cells are immune system cells selected fromthe group consisting of PBMCs, monocytes, macrophages, dendritic cells,lymphocytes and antigen-presenting cells; and c) evaluating an effect ofthe test agent on the diseased cells in the culture of b), therebyevaluating a test agent in an in vitro diseased cell model.
 3. A methodof evaluating a test agent in an in vitro diseased cell model, saidmethod comprising: a) priming a culture of epithelial and/or endothelialcells with a test agent; b) transferring cells from the culture of a) toa culture of a lymphoid tissue; c) transferring cells, culture media, orcells and culture media from the culture of b) to a culture of diseasedcells, wherein said diseased cells are immune system cells selected fromthe group consisting of PBMCs, monocytes, macrophages, dendritic cells,lymphocytes and antigen-presenting cells; and d) evaluating an effect ofthe test agent on the diseased cells in the culture of c), therebyevaluating a test agent in an in vitro diseased cell model.
 4. Themethod of claim 1, wherein said agent is selected from the groupconsisting of vaccines, adjuvants, immunotherapy candidates, cosmetics,drugs, biologics, and chemical compounds.
 5. The method of claim 2,wherein said agent is selected from the group consisting of vaccines,adjuvants, immunotherapy candidates, cosmetics, drugs, biologics, andchemical compounds.
 6. The method of claim 3, wherein said agent isselected from the group consisting of vaccines, adjuvants, immunotherapycandidates, cosmetics, drugs, biologics, and chemical compounds.
 7. Themethod of claim 1, wherein said diseased cells are selected from thegroup consisting of virally infected cells, bacterially infected cells,tumor cells, and autoimmune disease-afflicted cells.
 8. The method ofclaim 7, wherein said diseased cells are virally infected cells selectedfrom the group consisting of: (i) virally infected PBMCs derived from ablood donor infected with the virus, and (ii) virally infected PBMCsinfected in vitro with a virus and derived from a blood donor that isuninfected by the virus.
 9. The method of claim 7, wherein said diseasedcells are bacterially infected cells selected from the group consistingof: (i) bacterially infected PBMCs derived from a blood donor infectedwith the bacterium, and (ii) bacterially infected PBMCs infected invitro with a bacterium and derived from a blood donor that is uninfectedby the bacterium.
 10. The method of claim 2, wherein said diseased cellsare selected from the group consisting of virally infected cells,bacterially infected cells, tumor cells, and autoimmunedisease-afflicted cells.
 11. The method of claim 10, wherein saiddiseased cells are virally infected cells selected from the groupconsisting of: (i) virally infected PBMCs derived from a blood donorinfected with the virus, and (ii) virally infected PBMCs infected invitro with a virus and derived from a blood donor that is uninfected bythe virus.
 12. The method of claim 10, wherein said diseased cells arebacterially infected cells selected from the group consisting of: (i)bacterially infected PBMCs derived from a blood donor infected with thebacterium, and (ii) bacterially infected PBMCs infected in vitro with abacterium and derived from a blood donor that is uninfected by thebacterium.
 13. The method of claim 3, wherein said diseased cells areselected from the group consisting of virally infected cells,bacterially infected cells, tumor cells, and autoimmunedisease-afflicted cells.
 14. The method of claim 13, wherein saiddiseased cells are virally infected cells selected from the groupconsisting of: (i) virally infected PBMCs derived from a blood donorinfected with the virus, and (ii) virally infected PBMCs infected invitro with a virus and derived from a blood donor that is uninfected bythe virus.
 15. The method of claim 13, wherein said diseased cells arebacterially infected cells selected from the group consisting of: (i)bacterially infected PBMCs derived from a blood donor infected with thebacterium, and (ii) bacterially infected PBMCs infected in vitro with abacterium and derived from a blood donor that is uninfected by thebacterium.
 16. The method of claim 1, wherein said effect of the testagent is an effect on a parameter selected from the group consisting ofcellular growth rate, cell number, apoptosis, cellular maturation,bacterial replication, viral replication, antibody generation, cytokineproduction, chemokine production, and cellular marker expression. 17.The method of claim 2, wherein said effect of the test agent is aneffect on a parameter selected from the group consisting of cellulargrowth rate, cell number, apoptosis, cellular maturation, bacterialreplication, viral replication, antibody generation, cytokineproduction, chemokine production, and cellular marker expression. 18.The method of claim 3, wherein said effect of the test agent is aneffect on a parameter selected from the group consisting of cellulargrowth rate, cell number, apoptosis, cellular maturation, bacterialreplication, viral replication, antibody generation, cytokineproduction, chemokine production, and cellular marker expression. 19.The method of claim 1, wherein the epithelial and/or endothelial cellsare on a two- or three-dimensional matrix.
 20. The method of claim 19,wherein the matrix is selected from the group consisting of collagen, ahydrogel, PLA, PLGA, gelatin, hyaluronic acid, or a synthetic material,or combinations thereof.
 21. The method of claim 1, wherein the lymphoidtissue is on a two- or three-dimensional matrix.
 22. The method of claim21, wherein the matrix is selected from the group consisting ofcollagen, a hydrogel, PLA, PLGA, gelatin, hyaluronic acid, or asynthetic material, or combinations thereof.
 23. The method of claim 1,wherein the epithelial and/or endothelial cells are on a two- orthree-dimensional matrix and the lymphoid tissue is on a two- orthree-dimensional matrix.
 24. The method of claim 23, wherein the matrixis selected from the group consisting of collagen, a hydrogel, PLA,PLGA, gelatin, hyaluronic acid, or a synthetic material, or combinationsthereof.
 25. The method of claim 2, wherein the epithelial and/orendothelial cells are on a two- or three-dimensional matrix.
 26. Themethod of claim 25, wherein the matrix is selected from the groupconsisting of collagen, a hydrogel, PLA, PLGA, gelatin, hyaluronic acid,or a synthetic material, or combinations thereof.
 27. The method ofclaim 2, wherein the lymphoid tissue is on a two- or three-dimensionalmatrix.
 28. The method of claim 27, wherein the matrix is selected fromthe group consisting of collagen, a hydrogel, PLA, PLGA, gelatin,hyaluronic acid, or a synthetic material, or combinations thereof. 29.The method of claim 2, wherein the epithelial and/or endothelial cellsare on a two- or three-dimensional matrix and the lymphoid tissue is ona two- or three-dimensional matrix.
 30. The method of claim 29, whereinthe matrix is selected from the group consisting of collagen, ahydrogel, PLA, PLGA, gelatin, hyaluronic acid, or a synthetic material,or combinations thereof.
 31. The method of claim 3, wherein theepithelial and/or endothelial cells are on a two- or three-dimensionalmatrix.
 32. The method of claim 31, wherein the matrix is selected fromthe group consisting of collagen, a hydrogel, PLA, PLGA, gelatin,hyaluronic acid, or a synthetic material, or combinations thereof. 33.The method of claim 3, wherein the lymphoid tissue is on a two- orthree-dimensional matrix.
 34. The method of claim 33, wherein the matrixis selected from the group consisting of collagen, a hydrogel, PLA,PLGA, gelatin, hyaluronic acid, or a synthetic material, or combinationsthereof.
 35. The method of claim 3, wherein the epithelial and/orendothelial cells are on a two- or three-dimensional matrix and thelymphoid tissue is on a two- or three-dimensional matrix.
 36. The methodof claim 35, wherein the matrix is selected from the group consisting ofcollagen, a hydrogel, PLA, PLGA, gelatin, hyaluronic acid, or asynthetic material, or combinations thereof.